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Appendix 22
Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 1
Development of a bovine enterovirus-based vector that expresses multi-epitope of FMDV
Jong-Hyeon Park [email protected] FMDV lab, FMD Division Animal Plant and Fisheries Quarantine and Inspection Agency (QIA),
480 Anyang 6 dong, Anyang, Republic of Korea
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT
Conclusions and Recommendations • A recombinant BEV vectors carrying B and T cell epitopes of
FMDV were constructed and recovered as live viruses • BEV-based viral vector is able to deliver approximately 720 bps of foreign genes • The rBEVs show a proper growth without affecting of replication
kinetics and plaque sizes compared to the parent virus. • The rBEVs were detected and isolated in feces and serum samples
after administration to cattle. • The calves inoculated with rBEVs showed no clinical signs • The rBEVs show a possibility as live vaccine vector for FMD - Experiment for the immune and protective effect against FMDV is still undertaken.
- Expression of larger sized FMDV molecule with molecular adjuvant should be tried
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT
Objectives
• To verify the possibility of BEV as live and
delivery vaccine vector
• To investigate the multiplication and safety as
a live vaccine candidate for FMD in vivo
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT
Why use BEV-1 (LC R4 strain) as a vaccine vector?
• Safe to animals (isolated from healthy dairy cattle, Kunin, 1957) - BEV infections are ubiquitous and are normally without significant or severe clinical
symptoms • The same Picornaviridae family with FMDV
• Thermostable - maintenance of the cold chain is not always guaranteed from manufacturing to
delivery)
• pH-stable - can be administered orally
• Very wide tissue tropism for cell types in vitro • Replicable in cattle (or other animals) • Generating high titers in various cell lines.
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT
FMDV and BEV genome
P1
P2
P3
Poly(C)
L VP4 VP2 VP3 VP1 2A 2B
2C
3A 3B 3C
3D
FMDV VPg
VP4 VP2 VP3 VP1
2A 2B
2C
3A 3B 3C
3D BEV
VPg
(Kbps)
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT
Strategy for rBEV construction
Cloning of full-length cDNA of BEV and recovery
• BEV Type 1 strain LCR4 (ATCC® Number: VR-248TM) • Cloned into pBluescript II SK (+) vector
T7
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT
Appendix 22
Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 2
EGFP and synthetic FMDV epitopes
• EGFP for marker gene: 720 bps
• O/Andong/SKR/2010 (O-SEA topotype, Mya-98 lineage isolated from epidemic of Andong region, Korea in Nov. 2010)
• O1 Manisa (ME-SA topotype, PanAsian lineage, vaccine strain) - FMDV VP1 (a.a 141-160, 200-213)
- FMDV VP4 (a.a 20-34) - FMDV 3A (a.a 21-35) • T-helper epitope
- Pan-HLA-DR (PADRE): a universal DR-restricted T-helper epitope
(Hung CF et al. 2007; Alexander J et al. 1994)
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT
Construction of rBEV containing foreign genes 2A Cleavage site (TSY/G)
BEV_EGFP EGFP (720 bps)
VP4
VP2
VP3
VP1
EGFP
2A
2B
2C
3A
3B
3C
3D
(A)16
BEV_O-AD-multi-epi/ O-Manisa-multi-epi
VP4
VP2
VP3
VP1
2A
2B
2C
3A 3B
3C
3D
(A)16
2A Cleavage site
(TSY/G)
(GGSGG) linker
FMDV_VP1200-213
327 bps(109aa)
linker linker
2A Cleavage site
(TSY/G)
linker linker
VP420-34
PADRE FMDV_VP1141-160 3A21-35
Hisx6
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT
rBEV-EGFP
rBEV-AD rBEV-Manisa
12
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT
EGFP expression from rBEV-EGFP ( 6 hours after infection in MDBK cells)
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT
Indirect immunofluorescence assay
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT
Appendix 22
Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 3
Growth of rBEVs in MDBK cells
10 BEV-full clone
8
BEV-EGFP BEV-AnDong multiepi 6
BEV-Manisa multiepi
4
2
0
Hour post-infection
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT
Search for pre-existing antibody against BEV-1 type in
the field
Distribution of BEV-1 (LC-R4 strain) VN titers in Korea
60 50 40
BEV-1 Cattle
BEV-1 Pigs
(N=172) (N=160)
• Positive rate of cattle is 48.3%
30 20 10 0
<2 2 2.5 3
• Positive rate of pigs is 70.6%
3.5 4 4.5 5 5.5 6 6.5 7 >7.0 BEV antibody titers (Log2)
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT
Infection of rBEVs to calves
Virus titer Calf No.
Inoculation virus
Inoculation route (TCID50/ml)
1180 9362 0522 0564
rBEV-EGFP
rBEV-AnDong- Multi-epi
107.29
107.44
Intramuscular 2ml,
Intranasal 2ml,
Oral 2ml
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT
No clinical sign during 14 days after inoculation
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT
RT-PCR for detection of rBEVs from infected calves
BEV in feces samples (DPI) Groups
Calf No.
0
1
2
3
4
5
6
1180
-
+
-
-
-
+
+
rBEV-EGFP 9362
-
+
-
-
-
+
+
rBEV-AD
multi
0522 - + 0564 - +
- - - + + - - - + +
BEV in serum samples (DPI) Groups
Calf No.
0
1
2
3
4
5
6
rBEV-AD
1180
-
nd
-
nd
-
nd
+ multi
9362
-
nd
-
nd
-
nd
+ 0522
-
nd
-
nd
-
nd
+
rBEV-EGFP 0564
-
nd
-
nd
-
nd
+ nd : not done
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT
Virus isolation of rBEV in feces of inoculated calves Bright-field
Fluorescence
CPE
rBEV-EGFP
No CPE
CPE rBEV-AnDong
-Multi-epi
CPE
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT
Appendix 22
Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 4
Virus neutralizing antibody against BEV and FMDV
Serum Neutralization Titers using BEV-1 LCR4
Calf No.
Inoculation
virus
0dpi 2dpi 4dpi
6dpi 8dpi 10dpi 12dpi 13dpi
1180
<8
<8
<8
<8
<8
<8
<8
<8 BEV-EGFP
9362
<8
<8
<8
<8
<8
<8
<8
<8
0522
BEV-AD-
<8 <8 <8
<8 <8 <8 <8 <8
0564
multi-epi <8
<8 <8 <8
<8 <8 <8 <8
Serum Neutralization Titers using O/AnDong/SKR/2010
Calf No.
Inoculation
virus
0dpi 2dpi 8dpi
10dpi 12dpi 13dpi
1180
<8
<8
<8
<8
<8
<8 BEV-EGFP
9362
<8
<8
<8
<8
<8
<8
0522
BEV-AD-
<8 <8 <8
<8 <8 <8
0564
multi-epi
<8
<8 <8 <8 <8 22
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT
Conclusions and Recommendations • A recombinant BEV vectors carrying B and T cell epitopes of
FMDV were constructed and recovered as live viruses • BEV-based viral vector is able to deliver approximately 720 bps of foreign genes • The rBEVs show a proper growth without affecting of replication
kinetics and plaque sizes compared to the parent virus. • The rBEVs were detected and isolated in feces and serum samples
after administration to cattle. • The calves inoculated with rBEVs showed no clinical signs • The rBEVs show a possibility as live vaccine vector for FMD - Experiment for the immune and protective effect against FMDV is still undertaken.
- Expression of larger sized FMDV molecule with molecular adjuvant should be tried
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT
Acknowledgements
Members of FMDV Lab, FMD Division, QIA. Korea. Jia-Qi Chu, Jeong-Nam Park, Yeo-Joo Lee, Rae-Hyung Kim,
Su-Mi Kim, Kwang-Nyeong Lee,
Hyang-Sim Lee, Young-Joon Ko, Byounghan Kim,
Seo-Yong Lee, Soo-Jeong Jung
EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT